Building on past progress in defining the structure and stability of the resting form of lipoxygenases and the EPR spectroscopy of the activated, ferric form, the overall aim of the next period is to obtain a molecular understanding of the lipoxygenase mechanism. EPR programs written in the last period will be expanded to include ferrous-nitric oxide complexes and dispersion and rapid passage spectra. Spectra at X- and Q-bands will be analyzed to determine the contributions of D and E to distributions of parameters in observed spectra. Polycrystalline and single crystal studies of lipoxygenase by EPR will continue using crystals of the enzyme in the ferric form and the nitric- oxide-complexed ferrous form. These studies are designed to determine the orientation of exchangeable ligands on iron within the lipoxygenase structure and how the principle axes of the magnetic tensors are related to the ligands. Pursuant to the goal of a molecular understanding of the fatty acid binding cavity and how positional specificity is achieved, new approaches will be developed for defining fatty acid binding, including using photoreactive fatty acid analogs and EPR studies. The importance of a novel pi-helical region for lipoxygenase function will be tested. Single site mutations and deletions, designed to vary the way the pi-helix contributes ligands to iron will be prepared and will be examined for activity, iron content, EPR spectra and thermal stability. Further studies of the mechanism of lipoxygenase unfolding will include studies to test whether iron binding is reversible, to use a soybean isoform to examine the reversible step in unfolding, and to use a spin labeled form of the resting enzyme to test the effects of glassing agents on cold denaturation. The crystallography effort will be supported in two ways. SBL-1 will be provided for continued experiments involving co-crystallization of ferric and ferrous enzyme with inhibitors. Human platelet 12-LO will continue to be produced in Sf9 insect cells, conditions for producing and storing this lipoxygenase will be improved and the protein will be supplied for x-ray crystallography. Fe levels will be measured in 12-LO.